Crystalline forms of a biphenyl compound

ABSTRACT

The invention provides crystalline forms of biphenyl-2-ylcarbamic acid 1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester, and pharmaceutically acceptable solvates thereof. The crystalline form can be a freebase, or a salt such as a diphosphate, monosulfate or dioxalate salt. The invention also provides pharmaceutical compositions comprising these crystalline compounds or prepared using these compounds; processes and intermediates for preparing the crystalline compounds; and methods of using these compounds to treat a pulmonary disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/740,465, filed Jan.14, 2013, now allowed; which is a divisional of U.S. Ser. No.12/817,543, filed Jun. 17, 2010, now U.S. Pat. No. 8,377,965; which is acontinuation of U.S. Ser. No. 12/563,788, filed Sep. 21, 2009, nowabandoned; which is a continuation of U.S. Ser. No. 11/890,880, filedAug. 8, 2007, now U.S. Pat. No. 7,700,777; which is a continuation ofU.S. Ser. No. 11/371,445, filed Mar. 9, 2006, now abandoned; whichclaims the benefit of U.S. Provisional Application No. 60/660,208, filedon Mar. 10, 2005; the entire disclosures of which are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel crystalline forms of a biphenylcompound and solvates thereof, which are expected to be useful fortreating pulmonary disorders. The invention also relates topharmaceutical compositions comprising the crystalline compounds orprepared from such compounds, processes and intermediates for preparingsuch crystalline compounds and methods of using such compounds to treata pulmonary disorder.

2. State of the Art

Commonly-assigned U.S. Patent Publication No. 2005/0203133 to Mammen etal. discloses novel biphenyl compounds that are expected to be usefulfor treating pulmonary disorders such as chronic obstructive pulmonarydisease (COPD) and asthma. In particular, the compoundbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester is specifically described in this application as possessingmuscarinic receptor antagonist or anticholinergic activity.

The chemical structure of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester isrepresented by formula I:

The compound of formula I has been named using thecommercially-available AutoNom software (MDL, San Leandro, Calif.).

Therapeutic agents useful for treating pulmonary or respiratorydisorders are advantageously administered directly into the respiratorytract by inhalation. In this regard, several types of pharmaceuticalinhalation devices have been developed for administering therapeuticagents by inhalation including dry powder inhalers (DPI), metered-doseinhalers (MDI) and nebulizer inhalers. When preparing pharmaceuticalcompositions and formulations for use in such devices, it is highlydesirable to have a crystalline form of the therapeutic agent that isneither hygroscopic nor deliquescent and which has a relatively highmelting point (typically greater than about 150° C.) thereby allowingthe material to be micronized without significant decomposition.

No crystalline forms of the compound of formula I have been reportedpreviously. Accordingly, a need exists for a stable, non-deliquescentcrystalline forms of the compound of formula I which have acceptablelevels of hygroscopicity and relatively high melting points.

SUMMARY OF THE INVENTION

The present invention provides crystalline forms ofbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (formula I). The crystalline form can be a freebase, apharmaceutically acceptable salt such as a diphosphate, monosulfate ordioxalate salt, or a pharmaceutically acceptable solvate of such salt.

Surprisingly, crystalline forms of the invention have been found not tobe deliquescent, even when exposed to atmospheric moisture.Additionally, the crystalline forms of the invention have acceptablelevels of hygroscopicity and acceptable melting points, greater thanabout 70° C. For example, the diphosphate salt has a melting pointaround 150° C.

Among other uses, crystalline forms of the compound of formula I areuseful for preparing pharmaceutical compositions expected to haveutility in treating pulmonary disorders. Accordingly, one aspect of theinvention pertains to a pharmaceutical composition comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of a crystalline form of the compound of formula I.

Yet another aspect of the invention pertains to compositions comprisinga crystalline form of the compound of formula I in combination with oneor more other therapeutic agents. Accordingly, in one embodiment, theinvention is directed to a composition comprising (a) a pharmaceuticallyacceptable carrier and a therapeutically effective amount of acrystalline form of the compound of formula I; and (b) a therapeuticallyeffective amount of an agent selected from a steroidal anti-inflammatoryagent such as a corticosteroid; a β₂ adrenergic receptor agonist; aphosphodiesterase-4 inhibitor; or a combination thereof; wherein thecrystalline form and the agent are formulated together or separately.When the agent is formulated separately, a pharmaceutically acceptablecarrier may be included.

Another aspect of the invention relates to a pharmaceutical compositioncomprising an aqueous isotonic saline solution comprising a crystallineform of the compound of formula I, wherein the solution has a pH in therange of from about 4 to 6. In a particular embodiment, an aqueousnebulizer formulation is buffered with citrate buffer to a pH of about5. In another particular embodiment, the aqueous nebulizer formulationcontains about 0.5 mg/mL free base equivalents of biphenyl-2-ylcarbamicacid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester.

In one embodiment, this invention provides a drug delivery devicecomprising a dry powder inhaler containing a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a crystalline formof the compound of formula I.

The compound of formula I has muscarinic receptor antagonist activity.Accordingly, crystalline forms of the compound of formula I are usefulfor treating pulmonary disorders such as asthma and chronic obstructivepulmonary disease. Thus, another aspect of the invention pertains to amethod for treating a pulmonary disorder comprising administering to apatient a therapeutically effective amount of a crystalline form of thecompound of formula I. Still another aspect of the invention relates toa method of producing bronchodilation in a patient comprisingadministering to the patient a bronchodilation-producing amount of acrystalline form of the compound of formula I. In one embodiment, thecompound is administered by inhalation. The invention also provides amethod of treating chronic obstructive pulmonary disease or asthmacomprising administering to a patient a therapeutically effective amountof a crystalline form of the compound of formula I. Another aspect ofthe invention is directed to a method for antagonizing a muscarinicreceptor in a mammal comprising administering to the mammal atherapeutically effective amount of a crystalline form of the compoundof formula I.

The invention is also directed to processes for preparing crystallineforms of the compound of formula I. The invention also provides aprocess for purifying the compound of formula I comprising forming acrystalline salt or a crystalline freebase of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester. The invention is further directed to products prepared by theprocesses described herein.

The invention is also directed to a crystalline form of the compound offormula I in a micronized form; and to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a micronizedcrystalline form of the compound of formula I.

The invention is also directed to a crystalline form of the compound offormula I for use in therapy or as a medicament. Additionally, theinvention relates to the use of a crystalline form of the compound offormula I for the manufacture of a medicament; especially for themanufacture of a medicament for the treatment of a pulmonary disorder orfor antagonizing a muscarinic receptor in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of a crystallinediphosphate salt of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (the compound of formula I).

FIG. 2 shows a differential scanning calorimetry (DSC) trace for thiscrystalline salt.

FIG. 3 shows a thermal gravimetric analysis (TGA) trace for thiscrystalline salt.

FIG. 4 shows a dynamic moisture sorption (DMS) trace for thiscrystalline salt.

FIG. 5 is a micrographic image of this crystalline salt.

FIG. 6 and FIG. 7 show a PXRD pattern and a DSC trace, respectively, fora less stable form of a crystalline diphosphate salt.

FIG. 8 shows a PXRD pattern of a crystalline monosulfate salt of thecompound of formula I. This crystalline salt is further characterized bythe TGA trace in FIG. 9, the DSC trace in FIG. 10, the DMS trace in FIG.11, and the micrographic image in FIG. 12.

FIG. 13 shows a PXRD pattern of a crystalline dioxalate salt of thecompound of formula I. This crystalline salt is further characterized bythe TGA trace in FIG. 14, the DSC trace in FIG. 15, the DMS trace inFIG. 16, and the micrographic image in FIG. 17.

FIG. 18 shows a PXRD pattern of Form I of the crystalline freebase ofthe compound of formula I. This crystalline freebase is furthercharacterized by the DSC trace in FIG. 19, the TGA trace in FIG. 20, theDMS trace in FIG. 21, and the micrographic image in FIG. 22.

FIG. 23 shows a PXRD pattern of Form II of the crystalline freebase ofthe compound of formula I. This crystalline freebase is furthercharacterized by the DSC trace in FIG. 24, the TGA trace in FIG. 25, andthe DMS trace in FIG. 26.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides crystalline forms of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester. The crystalline form can be a freebase, a pharmaceuticallyacceptable salt such as a diphosphate, monosulfate or dioxalate salt, ora pharmaceutically acceptable solvate of such salt. In a particularembodiment, the crystalline form is a diphosphate salt.

DEFINITIONS

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a crystalline compound of formula I, and oneor more molecules of a solvent. Such solvates typically have asubstantially fixed molar ratio of solute and solvent. This term alsoincludes clathrates, including clathrates with water. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

The term “Form I” refers to the crystalline freebase that is prepared bya method that uses water as part of a solvent mixture as the inertdiluent. The term “Form II” refers to the crystalline freebase that isprepared by a method that uses an organic solvent mixture as the inertdiluent, i.e. no water.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.For example, a therapeutically effective amount for antagonizing amuscarinic receptor is that amount which will achieve the desiredantagonizing effect. Similarly, a therapeutically effective amount fortreating a pulmonary disorder is that amount that will achieve thedesired therapeutic result, which may be disease prevention,amelioration, suppression or alleviation, as described below.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patientsuch as a mammal (particularly a human) that includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient believed to be at risk        of contracting or being pre-disposed to such disease or medical        condition;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient having such disease or medical condition;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient having such disease or medical condition; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient having such disease or medical condition.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable. For example, the term“pharmaceutically acceptable carrier” refers to a material that can beincorporated into a composition and administered to a patient withoutcausing undesirable biological effects or interacting in a deleteriousmanner with other components of the composition. Such pharmaceuticallyacceptable materials typically have met the required standards oftoxicological and manufacturing testing, and include those materialsidentified as suitable inactive ingredients by the U.S. Food and Drugadministration.

The term “unit dosage form” refers to a physically discrete unitsuitable for dosing a patient, i.e., each unit containing apredetermined quantity of a compound of the invention calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like.

Synthesis

The crystalline compounds of the invention can be synthesized fromreadily available starting materials as described below and in theExamples. It will be appreciated that while specific process conditions(i.e. reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given, other process conditions can also be usedunless otherwise stated. Generally, the reactions are conducted in asuitable inert diluent, examples of which include, but are not limitedto, methanol, ethanol, isopropanol, isobutanol, ethyl acetate,acetonitrile, dichloromethane, methyl t-butyl ether, and the like, andmixtures thereof, typically containing water. Upon completion of any ofthe foregoing reactions, the crystalline compounds can be isolated fromthe reaction mixture by any conventional means such as precipitation,concentration, centrifugation and the like.

The biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester employed in the invention can be readily prepared fromcommercially available starting materials and reagents using theprocedures described in the Examples, or using the procedures describedin U.S. Patent Publication No. 2005/0203133 to Mammen et al.

The molar ratios described in the methods of the invention can bereadily determined by various methods available to those skilled in theart. For example, such molar ratios can be readily determined by ¹H NMR.Alternatively, elemental analysis and HPLC methods can be used todetermine the molar ratio.

Diphosphate Salt Crystal

A diphosphate salt of the invention typically contains between about 1.8and 2.2 molar equivalents of phosphate per molar equivalent of thecompound of formula I; including between about 1.9 and 2.1 molarequivalents of phosphate per molar equivalent of the compound of formulaI.

In general, a crystalline diphosphate salt of the compound of formula Ior a pharmaceutically acceptable solvate thereof can be prepared bycontacting biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester with phosphoric acid. For example, the ester can be contacted withdilute aqueous phosphoric acid to form an amorphous diphosphate salt,which is then contacted with an inert diluent.

To prepare the amorphous diphosphate salt, the ester is typicallydissolved in aqueous phosphoric acid, diluted with water and isolated bylyophilization. Generally, this reaction is conducted at a temperatureranging from about 0 to 30° C., such as about 24° C. The ratio ofmilligrams of the ester to microliters of 1M phosphoric acid is about1:3 to about 1:4, including about 1:3.5. The resulting amorphousdiphosphate salt is then typically contacted with about 15 mg/ml toabout 25 mg/ml of inert diluent. Generally, this reaction is conductedat a temperature ranging from about 50 to 70° C., such as about 60° C.

In a particular embodiment, 500 mg of the ester is taken up in 5 ml ofwater and 1.5 ml of 1M phosphoric acid. The pH is adjusted toapproximately pH 5.3 with additional 1M phosphoric acid (equaling 2.1molar equivalents). The clear solution is filtered, frozen andlyophilized to dryness to provide an amorphous diphosphate salt. Theresulting amorphous diphosphate salt is added to anisopropanol:acetonitrile (1:1) solution, followed by the addition ofwater. In this reaction, the ratio of milligrams of the amorphousdiphosphate salt to milliliters of isopropanol:acetonitrile is about2:0.9 to about 2:2, including about 2:1.

Alternatively, a crystalline diphosphate salt can be prepared bycontacting the ester with about 2.0 to about 2.1 molar equivalents ofphosphoric acid. Generally, this reaction is conducted in an inertdiluent at a temperature ranging from about 40 to 60° C., such as about50° C. In a particular embodiment, the ester is added to anisopropanol:acetonitrile (1:1) solution, followed by the addition ofwater. After heating, phosphoric acid is added. In this reaction, theratio of grams of the ester to milliliters of phosphoric acid is about5:14 to about 5:18, including about 5:16.

Both of the aforementioned processes for preparing crystallinediphosphate salts can lead to the formation of a separate, less stable,diphosphate crystal form. FIG. 6 and FIG. 7 show a PXRD pattern and aDSC trace, respectively, for this less stable form. The more stablediphosphate crystal is the prevalent form; however, when the less stablediphosphate form is present, it can be readily converted to the morestable crystal by increasing the water content in the solvent mixture,and reheating the suspension to about 50° C. to about 70° C., typicallyabout 60° C., for about 2 to about 6 hours, typically about 2 hours,followed by cooling to room temperature overnight with slow stirring.

Monosulfate Salt Crystal

A monosulfate salt of the invention typically contains between about 0.8and 1.2 molar equivalents of sulfate per molar equivalent of thecompound of formula I; including between about 0.9 and 1.1 molarequivalents of sulfate per molar equivalent of the compound of formulaI.

A crystalline monosulfate salt of the compound of formula I or apharmaceutically acceptable solvate thereof can be prepared bycontacting biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester with sulfuric acid. For example, the ester can be contacted with1N aqueous sulfuric acid to form a monosulfate salt, which is thencontacted with an inert diluent.

To prepare the monosulfate salt, the ester is typically dissolved in 1:1acetonitrile:water, diluted with aqueous sulfuric acid, diluted withwater and isolated by lyophilization. Generally, this reaction isconducted at a temperature ranging from about 0 to 30° C., such as about24° C. The ratio of milligrams of the ester to milliliters of 1Nsulfuric acid in water is about 325 mg/ml to about 285 mg/ml, includingabout 305 mg/ml. In one particular embodiment, 442 mg of the ester istaken up in 5 ml of 1:1 acetonitrile:water and 1.45 ml of 1N sulfuricacid is added slowly, while monitoring the pH. The pH is then adjustedto approximately pH 3.3. The clear solution is filtered, frozen andlyophilized to dryness to provide a monosulfate salt. The resultingmonosulfate salt is then typically contacted with about 10 mg/ml toabout 20 mg/ml of inert diluent. In one embodiment, this reaction isconducted at a first temperature and then at a lower second temperature,both temperatures ranging from about 50 to 80° C., such as about 60° C.to 70° C. In a particular embodiment, the monosulfate salt is added toan isopropanol:acetonitrile (10:1) solution. In this reaction, the ratioof milligrams of the monosulfate salt to milliliters ofisopropanol:acetonitrile is about 15:3 to about 15:0.8, including about15:1.

In another embodiment, this reaction is conducted at a first temperatureand then at two lower temperature cycles. The first temperature rangesfrom about 50 to 80° C., such as about 70° C. The first lowertemperature cycle varies from about 60° C. to 30° C. The second lowertemperature cycle varies from about 40° C. to 30° C. In a particularembodiment, the monosulfate salt is added to an isopropanol:acetonitrile(10:1) solution. In this reaction, the ratio of milligrams of themonohydrate salt to milliliters of isopropanol:acetonitrile is about161:7 to about 161:11, including about 161:9.

Dioxalate Salt Crystal

A dioxalate salt of the invention typically contains between about 1.8and 2.2 molar equivalents of oxalate per molar equivalent of thecompound of formula I; including between about 1.9 and 2.1 molarequivalents of oxalate per molar equivalent of the compound of formulaI.

A crystalline dioxalate salt of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester or a pharmaceutically acceptable solvate thereof, can be preparedby contacting biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester with oxalic acid. For example, the ester can be contacted with 1Maqueous oxalic acid to form a dioxalate salt, which is then contactedwith an inert diluent.

To prepare the dioxalate salt, the ester is typically dissolved in 1:1acetonitrile:water, diluted with aqueous oxalic acid, diluted with waterand isolated by lyophilization. Generally, this reaction is conducted ata temperature ranging from about 0 to 30° C., such as about 24° C. Theratio of milligrams of the ester to milliliters of 1M aqueous oxalicacid is about 320 mg/ml to about 280 mg/ml, including about 300 mg/ml.In one particular embodiment, 510 mg of the ester is taken up in 5 ml of1:1 acetonitrile:water and 1.7 ml of 1M aqueous oxalic acid is addedslowly, while monitoring the pH. The pH is adjusted to approximately pH3.0. The clear solution is filtered, frozen and lyophilized to drynessto provide a dioxalate salt.

In one embodiment, the resulting dioxalate salt is then typicallycontacted with about 5 mg/ml to about 15 mg/ml of inert diluent.Generally, this reaction is conducted at a temperature ranging fromabout 50 to 70° C., such as about 60° C. In a particular embodiment, thedioxalate salt is added to an isopropanol:water (94:6) solution. In thisreaction, the ratio of milligrams of the dioxalate salt to millilitersof isopropanol:water is about 10:0.8 to about 10:3, including about10:1.

A crystalline dioxalate salt can also be prepared by forming a seedcrystal of a crystalline dioxalate salt of the ester (synthesized asdescribed above), forming a dioxalate salt of the ester by contactingthe ester with oxalic acid and dissolving the salt in an inert diluentto form a solution, and adding the seed crystal to the solution.

In one embodiment, a dioxalate salt is typically contacted with about 5mg/ml to about 15 mg/ml of inert diluent. Generally, this reaction isconducted at a first temperature ranging from about 50 to 70° C., suchas about 60° C. The mixture is then cooled to a second temperatureranging from about 3 to 10° C., such as about 4° C. The seed crystal ofa crystalline dioxalate salt of the ester is then added. In a particularembodiment, the dioxalate salt is added to an isopropanol:water (94:6)solution. In this reaction, the ratio of milligrams of the dioxalatesalt to milliliters of isopropanol:water is about 150:10 to about150:16, including about 150:13.

Freebase Crystal

A crystalline freebase biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester or a pharmaceutically acceptable solvate thereof, can be preparedby contacting biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester with an inert diluent.

To prepare one form of a crystalline freebase (Form I), the ester istypically contacted with about 5 mg/ml to about 15 mg/ml of inertdiluent. Generally, this reaction is conducted at a temperature rangingfrom about 20 to 30° C., such as about 25° C. In a particularembodiment, the ester is added to a water:acetonitrile (1:1) solution.In this reaction, the ratio of milligrams of the ester to milliliters ofwater:acetonitrile is about 100:0.3 to about 100:1, including about100:0.5. Alternately, the reaction can be conducted at a firsttemperature ranging from about 20 to 30° C., such as about 25° C., andthen cooled to a second temperature ranging from about 3 to 10° C., suchas about 4° C.

A crystalline freebase can also be prepared by forming a seed crystal ofa crystalline freebase (synthesized as described above), forming acrystalline freebase by contacting the ester with an inert diluent anddissolving the resulting crystalline ester to form a solution, andadding the seed crystal to the solution.

In one embodiment, the ester is typically contacted with about 5 mg/mlto about 15 mg/ml of inert diluent. Generally, this reaction isconducted at a first temperature ranging from about 50 to 70° C., suchas about 60° C. The mixture is then cooled to a second temperatureranging from about 3 to 10° C., such as about 4° C. The seed crystal ofa crystalline freebase of the ester is then added, followed by severalheating and cooling cycles. The first heating cycle goes, for example,from about 30 to 40° C. and then to about 50° C., followed by cooling toroom temperature. The second, third and forth heating cycles involveheating the sample to a temperature ranging from about 50 to 70° C.,such as about 60° C., followed by cooling to room temperature. In aparticular embodiment, the ester is added to water:acetonitrile (1:1)solution, followed by the addition of water and further acetonitrile. Inthis reaction, the ratio of milligrams of the ester to milliliters ofacetonitrile and water is about 230:0.1 to about 230:0.5, includingabout 230:0.2.

To prepare another form of a crystalline freebase (Form II), the esteris typically contacted with about 200 mg/ml to about 100 mg/ml of inertdiluent. Generally, this reaction is conducted at a temperature rangingfrom about 20 to 30° C., such as about 25° C. A particularly suitableinert diluent is a combination of acetonitrile and methyl t-butyl ether.In a particular embodiment, biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester is added to acetonitrile, followed by the addition of methylt-butyl ether and additional acetonitrile. In this reaction, the ratioof milligrams of the ester to milliliters of acetonitrile:methyl t-butylether (1:2 solution) is about 200 mg/ml to about 100 mg/ml, includingabout 70:0.45.

Crystalline Properties

Among other advantages, it has been discovered that forming acrystalline compound of formula I is useful for purifying the compoundof formula I. For example: the crystalline diphosphate salt has a puritygreater than 96%, and typically greater than 98%.

As is well known in the field of powder x-ray diffraction (PXRD),relative peak heights of PXRD spectra are dependent on a number offactors relating to sample preparation and instrument geometry, whilepeak positions are relatively insensitive to experimental details. Thus,in one embodiment, the crystalline compounds of the invention arecharacterized by a PXRD pattern having certain peak positions.

In one embodiment, a crystalline diphosphate salt of the compound offormula I is characterized by a PXRD pattern having two or morediffraction peaks at 2θ values selected from 6.4±0.2, 7.6±0.2, 8.6±0.2,13.7±0.2, 15.0±0.2, 19.4±0.2, 21.6±0.2, 22.1±0.2, 22.9±0.2, and23.7±0.2. In one particular embodiment, this crystalline form ischaracterized by a PXRD pattern comprising diffraction peaks at 2θvalues of 15.0±0.2, 19.4±0.2, 21.6±0.2, and 23.7±0.2. In anotherembodiment, a crystalline diphosphate salt is characterized by a PXRDpattern in which the peak positions are substantially in accordance withthose shown in FIG. 1. Note the differences between the PXRD pattern inFIG. 1 and the PXRD pattern for the less stable diphosphate salt asdepicted in FIG. 6.

In one embodiment, a crystalline monosulfate salt of the compound offormula I is characterized by a PXRD pattern having two or morediffraction peaks at 2θ values selected from 7.7±0.2, 8.4±0.2, 8.8±0.2,12.6±0.2, 13.7±0.2, 14.1±0.2, 15.3±0.2, 16.0±0.2, 19.7±0.2, 20.6±0.2,23.0±0.2, and 24.4±0.2. In one particular embodiment, this crystallineform is characterized by a PXRD pattern comprising diffraction peaks at2θ values of 12.6±0.2, 19.7±0.2, 23.0±0.2, and 24.4±0.2. In anotherembodiment, a crystalline monosulfate salt is characterized by a PXRDpattern in which the peak positions are substantially in accordance withthose shown in FIG. 8.

In one embodiment, a crystalline dioxalate salt of the compound offormula I is characterized by a PXRD pattern having two or morediffraction peaks at 2θ values selected from 7.7±0.2, 8.7±0.2, 13.5±0.2,14.0±0.2, 14.8±0.2, 15.4±0.2, 15.8±0.2, 19.4±0.2, 22.9±0.2, 23.3±0.2,and 24.6±0.2. In one particular embodiment, this crystalline form ischaracterized by a PXRD pattern comprising diffraction peaks at 2θvalues of 8.7±0.2, 14.0±0.2, 19.4±0.2, and 22.9±0.2. In anotherembodiment, a crystalline dioxalate salt is characterized by a PXRDpattern in which the peak positions are substantially in accordance withthose shown in FIG. 13.

In one embodiment, a crystalline freebase (Form I) of the compound offormula I is characterized by a PXRD pattern having two or morediffraction peaks at 2θ values selected from 4.7±0.2, 9.6±0.2, 12.7±0.2,13.7±0.2, 16.7±0.2, 17.4±0.2, 18.5±0.2, 19.4±0.2, 20.8±0.2, 21.4±0.2,24.2±0.2, and 25.6±0.2. In one particular embodiment, this crystallineform is characterized by a PXRD pattern comprising diffraction peaks at2θ values of 4.7±0.2, 18.5±0.2, 20.8±0.2, and 25.6±0.2. In anotherembodiment, a crystalline freebase (Form I) is characterized by a PXRDpattern in which the peak positions are substantially in accordance withthose shown in FIG. 18.

In one embodiment, a crystalline freebase (Form II) of the compound offormula I is characterized by a PXRD pattern having two or morediffraction peaks at 2θ values selected from 4.6±0.2, 9.3±0.2, 12.9±0.2,13.6±0.2, 14.0±0.2, 14.6±0.2, 16.5±0.2, 18.6±0.2, 19.1±0.2, 20.9±0.2,22.1±0.2, 22.7±0.2, and 25.7±0.2. In one particular embodiment, thiscrystalline form is characterized by a PXRD pattern comprisingdiffraction peaks at 2θ values of 4.6±0.2, 18.6±0.2, 22.1±0.2, and22.7±0.2. In another embodiment, a crystalline freebase (Form II) ischaracterized by a PXRD pattern in which the peak positions aresubstantially in accordance with those shown in FIG. 23.

In yet another embodiment, the crystalline compounds of formula I arecharacterized by their differential scanning calorimetry (DSC) trace.Thus, a crystalline diphosphate salt of the compound of formula I ischaracterized by its DSC trace which showed a maximum endothermic heatflow at about 154.5° C., as illustrated in FIG. 2. Note the differencebetween the DSC trace in FIG. 2 and the DSC trace for the less stablediphosphate salt depicted in FIG. 7. The DSC trace for the stablecrystalline diphosphate salt (FIG. 2) shows a typical low temperaturetransition followed by a relatively sharp peak at about 154.5° C. On theother hand, the DSC trace for the unstable crystalline diphosphate salt(FIG. 7) shows a distinct shoulder prior to a much smaller meltingtransition at about 150.3° C.

Similarly, a crystalline monosulfate salt of the compound of formula Iis characterized by its DSC trace which showed a maximum endothermicheat flow at about 76.5° C., as illustrated in FIG. 10; a crystallinedioxalate salt is characterized by its DSC trace which showed a maximumendothermic heat flow at about 73.7° C., as illustrated in FIG. 15; acrystalline freebase (Form I) is characterized by its DSC trace whichshowed a maximum endothermic heat flow at about 102.7° C., asillustrated in FIG. 19; and a crystalline freebase (Form II) ischaracterized by its DSC trace which showed a maximum endothermic heatflow at about 98.6° C., as illustrated in FIG. 24.

The crystalline compounds of the invention have been demonstrated tohave a reversible sorption/desorption profile with an acceptable,moderate level of hygroscopicity: a crystalline diphosphate salt of thecompound of formula I exhibits less than 2% weight gain when exposed toup to 90% relative humidity; a crystalline monosulfate salt exhibitsless than 4% weight gain when exposed to up to 90% relative humidity; acrystalline dioxalate salt exhibits less than 3% weight gain whenexposed to up to 90% relative humidity; a crystalline freebase (Form I)exhibits less than 6% weight gain when exposed to up to 90% relativehumidity; and a crystalline freebase (Form II) exhibits less than 4%weight gain when exposed to up 90% relative humidity.

Additionally, the crystalline compounds of the invention have been foundto be stable upon exposure to elevated temperature and humidity. Forexample, after storage for 1 month at 40° C. and 75% relative humidity,analysis by high performance liquid chromatography (HPLC) showed nodetectable chemical degradation (i.e., less than 0.5% degradation) forthe crystalline compounds of the invention.

These properties of the crystalline compounds of the invention arefurther illustrated in the Examples below.

Pharmaceutical Compositions and Formulations

The crystalline compound of formula I is typically administered to apatient in the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration. However, it will be understood by those skilledin the art that, once the crystalline salt of the invention has beenformulated, it may no longer be in crystalline form, i.e., the salt maybe dissolved in a suitable carrier.

Accordingly, in one embodiment, the invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier or excipient and a crystalline biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester or a pharmaceutically acceptable solvate thereof. Thepharmaceutical composition may contain other therapeutic and/orformulating agents if desired.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a crystalline biphenyl-2-ylcarbamicacid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester or a pharmaceutically acceptable solvate thereof, as the activeagent. Typically, such pharmaceutical compositions will contain fromabout 0.01 to about 95% by weight of the active agent; including, fromabout 0.01 to about 30% by weight; such as from about 0.01 to about 10%by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combination of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials that can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols such aspropylene glycol; polyols such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; compressedpropellant gases such as chlorofluorocarbons and hydrofluorocarbons; andother non-toxic compatible substances employed in pharmaceuticalcompositions.

The pharmaceutical compositions of the invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills, canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of the invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a nebulizer inhaler. Such nebulizer devices typically produce astream of high velocity air that causes the pharmaceutical compositioncomprising the active agent to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Suitable nebulizer devices are commerciallyavailable, for example, by PARI GmbH (Starnberg, German). Othernebulizer devices include Respimat (Boehringer Ingelheim) and thosedescribed, for example, in U.S. Pat. No. 6,123,068 to Lloyd et al. andWO 97/12687 (Eicher et al.), the disclosures of which are incorporatedherein by reference in their entirety.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an aqueous solution comprising from about 0.05 μg/mLto about 10 mg/mL of a crystalline compound of formula I or apharmaceutically acceptable solvate thereof. In one embodiment, theaqueous formulation is isotonic. In one embodiment, the aqueousformulation has a pH in the range of from about 4 to 6. In a particularembodiment, the aqueous formulation is buffered with citrate buffer to apH of about 5. In another particular embodiment, the aqueous formulationcontains from about 0.1 mg/mL to about 1.0 mg/mL free base equivalentsof biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester.

In another specific embodiment of the invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a DPI. Such DPIs typically administer the active agent as afree-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free flowing powder, the active agentis typically formulated with a suitable excipient such as lactose orstarch. Micronization is a common method of reducing crystal size tothat suitable for pulmonary delivery. Typically, the active agent ismicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where “micronized particles” or“micronized form” means at least about 90% of the particles have adiameter of less than about 10 μm. Other methods of reducing particlesize may also be used such as fine milling, chopping, crushing,grinding, milling, screening, trituration, pulverization, and so forth,as long as the desired particle size can be obtained.

A representative pharmaceutical composition for use in a DPI comprisesdry lactose having a particle size between about 1 μm and about 100 μmand micronized particles of a crystalline compound of formula I, or apharmaceutically acceptable solvate thereof. Such a dry powderformulation can be made, for example, by combining the lactose with theactive agent and then dry blending the components. Alternatively, ifdesired, the active agent can be formulated without an excipient. Thepharmaceutical composition is then typically loaded into a dry powderdispenser, or into inhalation cartridges or capsules for use with a drypowder delivery device.

Examples of DPI delivery devices include Diskhaler (GlaxoSmithKline,Research Triangle Park, N.C.; see, e.g., U.S. Pat. No. 5,035,237 toNewell et al.); Diskus (GlaxoSmithKline; see, e.g., U.S. Pat. No.6,378,519 to Davies et al.); Turbuhaler (AstraZeneca, Wilmington, Del.;see, e.g., U.S. Pat. No. 4,524,769 to Wetterlin); Rotahaler(GlaxoSmithKline; see, e.g., U.S. Pat. No. 4,353,365 to Hallworth etal.) and Handihaler (Boehringer Ingelheim). Further examples of suitableDPI devices are described in U.S. Pat. No. 5,415,162 to Casper et al.,U.S. Pat. No. 5,239,993 to Evans, and U.S. Pat. No. 5,715,810 toArmstrong et al., and references cited therein. The disclosures of theaforementioned patents are incorporated herein by reference in theirentirety.

In yet another specific embodiment of the invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing an MDI, which typically discharges a measured amount of the activeagent or a pharmaceutically acceptable salt or solvate or stereoisomerthereof using compressed propellant gas. Accordingly, pharmaceuticalcompositions administered using an MDI typically comprise a solution orsuspension of the active agent in a liquefied propellant. Any suitableliquefied propellant may be employed including chlorofluorocarbons suchas CCl₃F, and hydrofluoroalkanes (HFAs) such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due to concerns aboutchlorofluorocarbons affecting the ozone layer, formulations containingHFAs are generally preferred. Additional optional components of HFAformulations include co-solvents such as ethanol or pentane, andsurfactants such as sorbitan trioleate, oleic acid, lecithin, andglycerin. See, for example, U.S. Pat. No. 5,225,183 to Purewal et al.,EP 0717987 A2 (Minnesota Mining and Manufacturing Company), and WO92/22286 (Minnesota Mining and Manufacturing Company, the disclosures ofwhich are incorporated herein by reference in their entirety.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01 to 5% by weight of a crystallinecompound of formula I, or a pharmaceutically acceptable solvate thereof;from about 0 to 20% by weight ethanol; and from about 0 to 5% by weightsurfactant; with the remainder being an HFA propellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing theactive agent, ethanol (if present) and the surfactant (if present). Toprepare a suspension, the active agent is micronized and then combinedwith the propellant. The formulation is then loaded into an aerosolcanister, which forms a portion of a metered-dose inhaler device.Examples of metered-dose inhaler devices developed specifically for usewith HFA propellants are described in U.S. Pat. No. 6,006,745 to Mareckiand U.S. Pat. No. 6,143,277 to Ashurst et al. Alternatively, asuspension formulation can be prepared by spray drying a coating ofsurfactant on micronized particles of the active agent. See, forexample, WO 99/53901 (Glaxo Group Ltd.) and WO 00/61108 (Glaxo GroupLtd.). The disclosures of the aforementioned patents and publicationsare incorporated herein by reference in their entirety.

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Pat. No. 6,268,533 to Gao et al., U.S. Pat. No. 5,983,956 to Trofast;U.S. Pat. No. 5,874,063 to Briggner et al.; and U.S. Pat. No. 6,221,398to Jakupovic et al.; and WO 99/55319 (Glaxo Group Ltd.) and WO 00/30614(AstraZeneca AB); the disclosures of which are incorporated herein byreference in their entirety.

In another embodiment, the pharmaceutical compositions of the inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a salt of theinvention as an active ingredient. The pharmaceutical composition may bepackaged in a unit dosage form.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise a crystalline compound of thepresent invention as the active ingredient and one or morepharmaceutically acceptable carriers such as sodium citrate or dicalciumphosphate. Optionally or alternatively, such solid dosage forms may alsocomprise: fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants such as glycerol; disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and/or sodium carbonate; solutionretarding agents such as paraffin; absorption accelerators such asquaternary ammonium compounds; wetting agents such as cetyl alcoholand/or glycerol monostearate; absorbents such as kaolin and/or bentoniteclay; lubricants such as talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and/or mixturesthereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically acceptable antioxidants include: water-solubleantioxidants such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; oil-solubleantioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA),butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like. Coating agents for tablets,capsules, pills and like, include those used for enteric coatings suchas cellulose acetate phthalate (CAP), polyvinyl acetate phthalate(PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the invention may also beformulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the invention mayoptionally contain opacifying agents and may be formulated so that theyrelease the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent such as,for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (especially cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

The crystalline compounds of the invention can also be administeredtransdermally using known transdermal delivery systems and excipients.For example, a compound of the invention can be admixed with permeationenhancers such as propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The crystalline compounds of the invention can also be co-administeredwith other therapeutic agents. This combination therapy involves using acompound of the invention combined with one or more of these secondaryagents, either formulated together (e.g., packaged together in a singleformulation) or formulated separately (e.g., packaged as separate unitdosage forms). Methods of formulating multiple agents together in thesame formulation or in separate unit dosage forms, are well known in theart.

The additional therapeutic agent(s) can be selected from otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents such as corticosteroids; non-steroidalanti-inflammatory agents (NSAIDs), and PDE₄ inhibitors); othermuscarinic receptor antagonists (i.e., antichlolinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibioticsor antivirals); antihistamines; protease inhibitors; and afferentblockers (e.g., D₂ agonists and neurokinin modulators).

One particular embodiment of the invention is directed to a compositioncomprising (a) a pharmaceutically acceptable carrier and atherapeutically effective amount of a crystalline form of the compoundof formula I; and (b) a pharmaceutically acceptable carrier and atherapeutically effective amount of an agent selected from a steroidalanti-inflammatory agent such as a corticosteroid; a β₂ adrenergicreceptor agonist; a phosphodiesterase-4 inhibitor; or a combinationthereof; wherein the compound of formula I and the agent are formulatedtogether or separately. In another embodiment, (b) is a pharmaceuticallyacceptable carrier and a therapeutically effective amount of a β₂adrenergic receptor agonist and a steroidal anti-inflammatory agent. Thesecondary agents can be used in the form of pharmaceutically acceptablesalts or solvates, and if appropriate, as optically pure stereoisomers.

Representative β₂ adrenergic receptor agonists that can be used incombination with crystalline compounds of the invention include, but arenot limited to, salmeterol, salbutamol, formoterol, salmefamol,fenoterol, terbutaline, albuterol, isoetharine, metaproterenol,bitolterol, pirbuterol, levalbuterol and the like, or pharmaceuticallyacceptable salts thereof. Other β₂ adrenergic receptor agonists that canbe used include, but are not limited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds described in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds described in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamideand related compounds described in WO 02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds described in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds described in U.S. Pat. No. 6,576,793 to Moran etal.; N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds described in U.S. Pat. No. 6,653,323 to Moran etal.; and pharmaceutically acceptable salts thereof. In a particularembodiment, the β₂-adrenoreceptor agonist is a crystallinemonohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.When employed, the β₂-adrenoreceptor agonist will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the β₂-adrenoreceptor agonist will be present in an amountsufficient to provide from about 0.05 μg to 500 μg per dose. Thedisclosures of the aforementioned patents and publications areincorporated herein by reference in their entirety.

Representative steroidal anti-inflammatory agents that can be used incombination with crystalline compounds of the invention include, but arenot limited to, methyl prednisolone, prednisolone, dexamethasone,fluticasone propionate, 6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydrofuran-3S-yl)ester, beclomethasone esters (e.g.,the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g., the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,ST-126 and the like, or pharmaceutically-acceptable salts thereof. Whenemployed, the steroidal anti-inflammatory agent will be present in thecomposition in a therapeutically effective amount. Typically, thesteroidal anti-inflammatory agent will be present in an amountsufficient to provide from about 0.05 μg to 500 μg per dose.

An exemplary combination is a crystalline form of the compound offormula I or solvate thereof, co-administered with salmeterol as the β₂adrenergic receptor agonist, and fluticasone propionate as the steroidalanti-inflammatory agent. Another exemplary combination is a crystallineform of the compound of formula I or solvate thereof, co-administeredwith a crystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent. Asnoted above, these agents can be formulated together or separately.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (e.g., sodium cromoglycate,nedocromil sodium, and phosphodiesterase (PDE) inhibitors such astheophylline, PDE4 inhibitors and mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

Representative phosphodiesterase-4 (PDE4) inhibitors or mixed PDE3/PDE4inhibitors that can be used in combination with the crystallinecompounds of the invention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds described inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds described in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with the crystalline compounds of theinvention include, but are not limited to, atropine, atropine sulfate,atropine oxide, methylatropine nitrate, homatropine hydrobromide,hyoscyamine (d,l) hydrobromide, scopolamine hydrobromide, ipratropiumbromide, oxitropium bromide, tiotropium bromide, methantheline,propantheline bromide, anisotropine methyl bromide, clidinium bromide,copyrrolate (Robinul), isopropamide iodide, mepenzolate bromide,tridihexethyl chloride (Pathilone), hexocyclium methylsulfate,cyclopentolate hydrochloride, tropicamide, trihexyphenidylhydrochloride, pirenzepine, telenzepine, AF-DX 116 and methoctramine andthe like, or a pharmaceutically acceptable salt thereof; or, for thosecompounds listed as a salt, alternate pharmaceutically acceptable saltthereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with the crystalline compounds of the inventioninclude, but are not limited to, ethanolamines such as carbinoxaminemaleate, clemastine fumarate, diphenylhydramine hydrochloride anddimenhydrinate; ethylenediamines such as pyrilamine amleate,tripelennamine hydrochloride and tripelennamine citrate; alkylaminessuch as chlorpheniramine and acrivastine; piperazines such ashydroxyzine hydrochloride, hydroxyzine pamoate, cyclizine hydrochloride,cyclizine lactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Unless otherwise indicated, exemplary suitable doses for the othertherapeutic agents administered in combination with a crystallinecompound of the invention are in the range of about 0.05 μg/day to 100mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the invention, as well as exemplary methods ofpreparation. One or more secondary agents can optionally be formulatedwith the crystalline compound of the invention (primary active agent).Alternately, the secondary agents(s) can be formulated separately andco-administered with the primary active agent, either simultaneously orsequentially. For example, in one embodiment, a single dry powderformulation can be manufactured to include both the crystalline compoundof the invention and one or more secondary agents. In anotherembodiment, one formulation is manufactured to contain the crystallinecompound of the invention and separate formulation(s) are manufacturedto contain the secondary agent(s). Such dry powder formulations can thenbe packaged in separate blister packs and administered with a single DPIdevice.

Exemplary Thy Powder Formulation for Administration by Inhalation

0.2 mg of a crystalline compound of the invention is micronized and thenblended with 25 mg of lactose. The blended mixture is then loaded into agelatin inhalation cartridge. The contents of the cartridge areadministered using a powder inhaler.

Exemplary Thy Powder Formulation for Administration by a Dry PowderInhaler

A dry powder is prepared having a bulk formulation ratio of micronizedcrystalline compound of the invention (active agent) to lactose of1:200. The powder is packed into a dry powder inhalation device capableof delivering between about 10 μg and 100 μg of active agent per dose.

Exemplary Formulations for Administration by a Metered Dose Inhaler

A suspension containing 5 wt % of a crystalline compound of theinvention (active agent) and 0.1 wt % lecithin is prepared by dispersing10 g of the active agent as micronized particles with a mean size lessthan 10 μm in a solution formed from 0.2 g of lecithin dissolved in 200mL of demineralized water. The suspension is spray dried and theresulting material is micronized to particles having a mean diameterless than 1.5 μm. The particles are loaded into cartridges withpressurized 1,1,1,2-tetrafluoroethane.

Alternately, a suspension containing 5 wt % of the active agent, 0.5 wt% lecithin, and 0.5 wt % trehalose is prepared by dispersing 5 g of theactive agent as micronized particles with a mean size less than 10 μm ina colloidal solution formed from 0.5 g of trehalose and 0.5 g oflecithin dissolved in 100 mL of demineralized water. The suspension isspray dried and the resulting material is micronized to particles havinga mean diameter less than 1.5 μm. The particles are loaded intocanisters with pressurized 1,1,1,2-tetrafluoroethane.

Exemplary Aqueous Aerosol Formulation for Administration by Nebulizer

A pharmaceutical composition is prepared by dissolving 0.5 mg of acrystalline compound of the invention (active agent) in 1 mL of a 0.9%sodium chloride solution acidified with citric acid. The mixture isstirred and sonicated until the active agent is dissolved. The pH of thesolution is adjusted to a value in the range of from 3 to 8 (typicallyabout 5) by the slow addition of NaOH.

Exemplary Hard Gelatin Capsule Formulation for Oral Administration

The following ingredients are thoroughly blended and then loaded into ahard gelatin capsule: 250 mg of a crystalline compound of the invention,200 mg of lactose (spray-dried), and 10 mg of magnesium stearate, for atotal of 460 mg of composition per capsule.

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of active ingredient per 10 mL of suspension.

Ingredients Amount Crystalline compound of the invention 1.0 g Fumaricacid 0.5 g Sodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben0.05 g Granulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

Exemplary Injectable Formulation

The following ingredients are blended and the pH is adjusted to 4±0.5using 0.5 N HCl or 0.5 N NaOH.

Ingredients Amount Crystalline compound of the invention 0.2 g Sodiumacetate buffer solution (0.4M) 2.0 mL HCl (0.5N) or NaOH (0.5N) q.s. topH 4 Water (distilled, sterile) q.s. to 20 mL

Utility

The compound of formula I possesses muscarinic receptor antagonistactivity and therefore, the crystalline form of the compound of formulaI is expected to be useful for treating medical conditions mediated bymuscarinic receptors, i.e., medical conditions that are ameliorated bytreatment with a muscarinic receptor antagonist. Such medical conditionsinclude, by way of example, pulmonary disorders or diseases includingthose associated with reversible airway obstruction such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis, allergic rhinitis, rhinorrhea,and the like. Other medical conditions that can be treated withmuscarinic receptor antagonists are genitourinary tract disorders suchas overactive bladder or detrusor hyperactivity and their symptoms;gastrointestinal tract disorders such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; cardiac arrhythmias such as sinus bradycardia;Parkinson's disease; cognitive disorders such as Alzheimer's disease;dismenorrhea; and the like.

Accordingly, in one embodiment, the invention is directed to a methodfor treating a pulmonary disorder, the method comprising administeringto a patient a therapeutically effective amount of a crystallinebiphenyl-2-ylcarbamic acid 1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester or apharmaceutically acceptable solvate thereof. When used to treat apulmonary disorder, the crystalline compound of the invention willtypically be administered by inhalation in multiple doses per day, in asingle daily dose or a single weekly dose. Generally, the dose fortreating a pulmonary disorder will range from about 10 μg/day to 200μg/day.

When administered by inhalation, the crystalline compounds of theinvention typically have the effect of producing bronchodilation.Accordingly, in another embodiment, the invention is directed to amethod of producing bronchodilation in a patient, the method comprisingadministering to a patient a bronchodilation-producing amount of acrystalline biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester or a pharmaceutically acceptable solvate thereof. Generally, thetherapeutically effective dose for producing bronchodilation will rangefrom about 10 μg/day to 200 μg/day.

In one embodiment, the invention is directed to a method of treatingchronic obstructive pulmonary disease or asthma, the method comprisingadministering to a patient a therapeutically effective amount of acrystalline biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester or a pharmaceutically acceptable solvate thereof. When used totreat a COPD or asthma, the salt of the invention will typically beadministered by inhalation in multiple doses per day or in a singledaily dose. Generally, the dose for treating COPD or asthma will rangefrom about 10 μg/day to 200 μg/day. As used herein, COPD includeschronic obstructive bronchitis and emphysema (see, for example, Barnes,Chronic Obstructive Pulmonary Disease, N Engl J Med 343:269-78 (2000)).

When used to treat a pulmonary disorder, the crystalline compounds ofthe invention are optionally administered in combination with othertherapeutic agents. Accordingly, in a particular embodiment, thepharmaceutical compositions and methods of the invention furthercomprise a therapeutically effective amount of a β₂-adrenoreceptoragonist, a corticosteroid, a non-steroidal anti-inflammatory agent, orcombination thereof.

In another embodiment, the crystalline compounds of the invention areused to antagonize a muscarinic receptor in biological system, and amammal in particular such as mice, rats, guinea pigs, rabbits, dogs,pigs, humans and so forth. In this embodiment, a therapeuticallyeffective amount of a crystalline compound of formula I is administeredto the mammal. If desired, the effects of antagonizing the muscarinicreceptor can then determined using conventional procedures andequipment.

The properties and utility of the crystalline compounds of the inventioncan be demonstrated using various in vitro and in vivo assays that arewell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated. The following abbreviations have thefollowing meanings unless otherwise indicated and any otherabbreviations used herein and not defined have their standard meaning:

-   -   AC adenylyl cyclase    -   ACh acetylcholine    -   ACN acetonitrile    -   BSA bovine serum albumin    -   cAMP 3′-5′ cyclic adenosine monophosphate    -   CHO Chinese hamster ovary    -   cM₅ cloned chimpanzee M5 receptor    -   DCM dichloromethane (i.e., methylene chloride)    -   DIPEA N,N-diisopropylethylamine    -   dPBS Dulbecco's phosphate buffered saline    -   DMSO dimethyl sulfoxide    -   EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide    -   EDTA ethylenediaminetetraacetic acid    -   FBS fetal bovine serum    -   FLIPR fluorometric imaging plate reader    -   HBSS Hank's buffered salt solution    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   hM₁ cloned human M1 receptor    -   hM₂ cloned human M2 receptor    -   hM₃ cloned human M3 receptor    -   hM₄ cloned human M4 receptor    -   hM₅ cloned human M5 receptor    -   HOBT N-hydroxybenzotriazole    -   HPLC high-performance liquid chromatography    -   IPA isopropanol    -   MCh methylcholine    -   MTBE methyl t-butyl ether    -   TFA trifluoroacetic acid

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, reagents, startingmaterials and solvents were purchased from commercial suppliers (such asSigma-Aldrich, Fluka, and the like) and were used without furtherpurification.

Unless otherwise indicated, HPLC analysis was conducted using an Agilent(Palo Alto, Calif.) Series 1100 instrument equipped with a Zorbax BonusRP 2.1×50 mm column (Agilent) having a 3.5 micron particle size.Detection was by UV absorbance at 214 nm. The mobile phases employedwere as follows (by volume): A is ACN (2%), water (98%) and TFA (0.1%);and B is acetonitrile (90%), water (10%) and TFA (0.1%). HPLC 10-70 datawas obtained using a flow rate of 0.5 mL/minute of 10 to 70% B over a 6minute gradient (with the remainder being A). Similarly, HPLC 5-35 dataand HPLC 10-90 data were obtained using 5 to 35% B; or 10 to 90% B overa 5 minute gradient.

Liquid chromatography mass spectrometry (LCMS) data were obtained withan Applied Biosystems (Foster City, Calif.) Model API-150EX instrument.LCMS 10-90 data was obtained using 10 to 90% Mobile Phase B over a 5minute gradient.

Preparation 1 Biphenyl-2-ylcarbamic Acid Piperidin-4-yl Ester

Biphenyl-2-isocyanate (97.5 g, 521 mmol) and4-hydroxy-N-benzylpiperidine (105 g, 549 mmol) were heated together at70° C. for 12 hours. The reaction mixture was then cooled to 50° C. andethanol (1 L) was added and then 6M HCl (191 mL) was added slowly. Theresulting mixture was then cooled to ambient temperature and ammoniumformate (98.5 g, 1.56 mol) was added and then nitrogen gas was bubbledthrough the solution vigorously for 20 minutes. Palladium on activatedcarbon (20 g, 10 wt % dry basis) was then added and the reaction mixturewas heated at 40° C. for 12 hours, and then filtered through a pad ofCelite. The solvent was then removed under reduced pressure and 1M HCl(40 mL) was added to the crude residue. The pH of the mixture was thenadjusted with 10 N NaOH to pH 12. The aqueous layer was extracted withethyl acetate (2×150 mL) and the organic layer was dried (magnesiumsulfate), filtered and the solvent removed under reduced pressure togive 155 g of the title intermediate (100% yield). HPLC (10-70)R_(t)=2.52; m/z: [M+H⁺] calc'd for C₁₈H₂₀N₂O₂ 297.15. found 297.3.

Preparation 2 N-Benzyl-N-methylaminoacetaldehyde

To a 3-necked 2-L flask was added N-benzyl-N-methylethanolamine (30.5 g,0.182 mol), DCM (0.5 L), DIPEA (95 mL, 0.546 mol) and DMSO (41 mL, 0.728mol). Using an ice bath, the mixture was cooled to about −10° C. andsulfur trioxide pyridine-complex (87 g, 0.546 mol) was added in 4portions over 5 minute intervals. The reaction was stirred at −10° C.for 2 hours. Before removing the ice-bath, the reaction was quenched byadding water (0.5 L). The aqueous layer was separated and the organiclayer was washed with water (0.5 L) and brine (0.5 L) and then driedover magnesium sulfate and filtered to provide the title compound whichwas used without further purification.

Preparation 3 Biphenyl-2-ylcarbamic Acid1-[2-(Benzylmethylamino)ethyl]piperidin-4-yl Ester

To a 2-L flask, containing the product of Preparation 2 in DCM (0.5 L)was added the product of Preparation 1 (30 g, 0.101 mol) followed bysodium triacetoxyborohydride (45 g, 0.202 mol). The reaction mixture wasstirred overnight and then quenched by the addition of 1 N hydrochloricacid (0.5 L) with vigorous stirring. Three layers were observed and theaqueous layer was removed. After washing with 1N NaOH (0.5 L), ahomogenous organic layer was obtained which was then washed with asaturated solution of aqueous NaCl (0.5 L), dried over magnesiumsulfate, filtered and the solvent removed under reduced pressure. Theresidue was purified by dissolving it in a minimal amount of isopropanoland cooling this solution to 0° C. to form a solid which was collectedand washed with cool isopropanol to provide 42.6 g of the title compound(95% yield). MS m/z: [M+H⁺] calc'd f for C₂₈H₃₃N₃O₂ 444.3. found 444.6.R_(f)=3.51 min (10-70 ACN:H₂O, reverse phase HPLC).

Preparation 3A Biphenyl-2-ylcarbamic Acid1-[2-(Benzylmethylamino)ethyl]piperidin-4-yl Ester

The title compound was prepared by mesylation of N-benzyl-N-methylethanolamine, which was then reacted with biphenyl-2-ylcarbamic acidpiperidin-4-yl ester in an alkylation reaction.

A 500 mL flask (reactor flask) was charged withN-benzyl-N-methylethanolamine (24.5 mL), DCM (120 mL), NaOH (80 mL; 30wt %) and tetrabutylammonium chloride. Mixing at low speed throughoutthe reaction, the mixture was cooled to −10° C. (cooling bath), and theaddition funnel charged with DCM (30 mL) and mesyl chloride (15.85 mL),which was added drop wise at a constant rate over 30 minutes. Theaddition was exothermic, and stirring was continued for 15 minutes whilethe temperature equilibrated back to −10° C. The reaction was held forat least 10 minutes to ensure full hydrolysis of the excess mesylchloride.

A 250 mL flask was charged with biphenyl-2-ylcarbamic acidpiperidin-4-yl ester (26 g; prepared as described in Preparation 1) andDCM (125 mL), stirred for 15 minutes at room temperature, and themixture chilled briefly to 10° C. to form a slurry. The slurry was thencharged into the reactor flask via the addition funnel. The cooling bathwas removed and the reaction mixture was warmed to 5° C. The mixture wastransferred to a separatory funnel, the layers allowed to settle, andthe aqueous layer removed. The organic layer was transferred back to thereactor flask, stirring resumed, the mixture held to room temperature,and the reaction monitored by HPLC for a total of 3.5 hours.

The reactor flask was charged with NaOH (1M solution; 100 mL), stirred,and the layers allowed to settle. The organic layer was separated,washed (NaCl satd. solution), its volume partially reduced under vacuum,and subjected to repeated IPA washings. The solids were collected andallowed to air-dry (25.85 g, 98% purity). Additional solids wereobtained from further processing of the mother liquor (volume reduction,IPA, cooling).

Preparation 4 Biphenyl-2-ylcarbamic Acid1-(2-Methylaminoethyl)piperidin-4-yl Ester

To a Parr hydrogenation flask was added the product of Preparation 3 (40g, 0.09 mol) and ethanol (0.5 L). The flask was flushed with nitrogengas and palladium on activated carbon (15 g, 10 wt % (dry basis), 37%wt/wt) was added along with acetic acid (20 mL). The mixture was kept onthe Parr hydrogenator under a hydrogen atmosphere (˜50 psi) for 3 hours.The mixture was then filtered and washed with ethanol. The filtrate wascondensed and the residue was dissolved in a minimal amount of DCM.Isopropyl acetate (10 volumes) was added slowly to form a solid whichwas collected to provide 22.0 g of the title compound (70% yield). MSm/z: [M+H⁺] calc'd for C₂₁H₂₇N₃O₂ 354.2. found 354.3. R_(f)=2.96 min(10-70 ACN:H₂O, reverse phase HPLC).

Preparation 5 Biphenyl-2-ylcarbamic Acid1-{2-[(4-Formylbenzoyl)methylamino]ethyl}piperidin-4-yl Ester

To a three-necked 1-L flask was added 4-carboxybenzaldehyde (4.77 g,31.8 mmol), EDC (6.64 g, 34.7 mmol), HOBT (1.91 g, 31.8 mmol), and DCM(200 mL). When the mixture was homogenous, a solution of the product ofPreparation 4 (10 g, 31.8 mmol) in DCM (100 mL) was added slowly. Thereaction mixture was stirred at room temperature for approximately 16hours and then washed with water (1×100 mL), 1N HCl (5×60 mL), 1N NaOH(1×100 mL) brine (1×50 mL), dried over sodium sulfate, filtered andconcentrated to afford 12.6 g of the title compound (92% yield; 85%purity based on HPLC). MS m/z: [M+H⁺] calc'd for C₂₉H₃₁N₃O₄ 486.2. found486.4. R_(f)=3.12 min (10-70 ACN:H₂O, reverse phase HPLC).

Example 1 Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

To a three-necked 2-L flask was added isonipecotamide (5.99 g, 40.0mmol), acetic acid (2.57 mL), sodium sulfate (6.44 g) and isopropanol(400 mL). The reaction mixture was cooled to 0-10° C. with an ice bathand a solution of biphenyl-2-ylcarbamic acid1-{2-[(4-formylbenzoyl)methylamino]ethyl}piperidin-4-yl ester (11 g,22.7 mmol; prepared as described in Preparation 5) in isopropanol (300mL) was slowly added. The reaction mixture was stirred at roomtemperature for 2 hours and then cooled to 0-10° C. Sodiumtriacetoxyborohydride (15.16 g, 68.5 mmol) was added portion wise andthis mixture was stirred at room temperature for 16 hours. The reactionmixture was then concentrated under reduced pressure to a volume ofabout 50 mL and this mixture was acidified with 1N HCl (200 mL) to pH 3.The resulting mixture was stirred at room temperature for 1 hour andthen extracted with DCM (3×250 mL). The aqueous phase was then cooled to0-5° C. with an ice bath and 50% aqueous NaOH solution was added toadjust the pH of the mixture to 10. This mixture was then extracted withisopropyl acetate (3×300 mL) and the combined organic layers were washedwith water (100 mL), brine (2×50 mL), dried over sodium sulfate,filtered and concentrated to afford 10.8 g of the title compound (80%yield. MS m/z: [M+H⁺] calc'd for C₃₅H₄₃N₅O₄ 598.3. found 598.6.R_(f)=2.32 min (10-70 ACN:H₂O, reverse phase HPLC).

Example 2 Crystalline Diphosphate Salt of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

500 mg of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (0.826 mmol of 96% pure material; prepared as described inExample 1) was taken up in 5 ml of water and 1.5 ml of 1M phosphoricacid. The pH was adjusted to approximately pH 5.3 with an additional0.25 ml of 1M phosphoric acid (equaling 2.1 molar equivalents). Theclear solution was filtered through a 0.2 micron filter, frozen andlyophilized to dryness to yield an amorphous diphosphate salt.

20 mg of the amorphous diphosphate salt was dissolved in 2 ml of IPA:ACN(1:1). 0.1 ml of water was added and the mixture heated to 60° C. understirring. Almost all of the solids dissolved. The suspension was allowedto cool to ambient temperature, under stirring, overnight. The resultingcrystals were collected by filtration and air-dried for 20 minutes togive the title compound (18.5 mg, 93% yield) as a white crystallinesolid. When examined under a microscope using polarized light, thecrystals exhibited some birefringence.

Example 3 Crystalline Diphosphate Salt of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

5.0 g of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (freebase; prepared as described in Example 1) was combined with80 ml of IPA:ACN (1:1). 4.0 ml of water was added and the mixture heatedto 50° C. under stirring, forming a clear solution. To this was addeddropwise at 50° C., 16 ml 1M phosphoric acid. The resulting cloudysolution was stirred at 50° C. for 5 hours, then allowed to cool toambient temperature, under slow stirring, overnight. The resultingcrystals were collected by filtration and air-dried for 1 hour, thenunder vacuum for 18 hours, to give the title compound (5.8 g, 75% yield)as a white crystalline solid (98.3% purity by HPLC).

Example 4 Crystalline Monosulfate Salt of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

442 mg of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (0.739 mmol of 96% pure material; prepared as described inExample 1) was taken up in 5 ml of H₂O:ACN (1:1) and 1.45 ml of 1Nsulfuric acid was added slowly, while monitoring the pH. The pH wasadjusted to approx. pH 3.3. The clear solution was filtered through a0.2 micron filter, frozen and lyophilized to dryness to yield amonosulfate salt.

30.3 mg of the monosulfate salt was dissolved in 1.65 ml of IPA:ACN(10:1). The suspension was heated by placing the vial in a pre-heated60° C. water bath for 30 minutes. A viscous material was formed and theheat increased to 70° C. for 30 minutes. Since the material remainedviscous, the heat was lowered to 60° C. and the mixture heated for anadditional hour. The heat was turned off and the mixture was allowed tocool to room temperature. After 4 days, the material appeared to besolid, and the sample was allowed to sit for an additional nine days.The solid was then filtered and dried using a vacuum pump for 1 hour togive the title compound (23 mg, 76% yield).

Example 5 Crystalline Monosulfate Salt of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

161 g of the monosulfate salt (prepared as described in Example 4) wasdissolved in 8.77 ml of IPA:ACN (10:1). The suspension was heated byplacing the vial in a pre-heated 70° C. water bath for 1.5 hours. Oildroplets formed within 5 minutes. The heat was lowered to 60° C. and themixture heated for an additional 1.5 hours, followed by heating at 50°C. for 40 minutes, at 40° C. for 40 minutes, then at 30° C. for 45minutes. The heat was turned off and the mixture was allowed to slowlycool to room temperature. The next day, the material was viewed under amicroscope and indicated needles and plates. The material was thenheated at 40° C. for 2 hours, at 35° C. for 30 minutes, and then at 30°C. for 30 minutes. The heat was turned off and the mixture was allowedto slowly cool to room temperature. The solid was then filtered anddried using a vacuum pump for 1 hour to give the title compound (117 mg,73% yield).

Example 6 Crystalline Dioxalate Salt of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

510 mg of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (0.853 mmol of 96% pure material; prepared as described inExample 1) was taken up in 5 ml of H₂O:ACN (1:1) and 1.7 ml of 1Maqueous oxalic acid was added slowly, while monitoring the pH. The pHwas adjusted to approx. pH 3.0. The clear solution was filtered througha 0.2 micron filter, frozen and lyophilized to dryness to yield adioxalate salt.

31.5 mg of the dioxalate salt was dissolved in 2.76 ml of 94% IPA/6%H₂O. The mixture was stirred in a pre-heated 60° C. water bath for 2.5hours. After 25 minutes, all of the sample was in solution. The heat wasturned off and the mixture was allowed to cool to room temperature. Thenext day, a small amount of viscous material was present. The vial wasrefrigerated at 4° C. After 4 days, the viscous material was stillpresent. The vial was then placed at room temperature and observed onemonth later. The material appeared to be solid, and was observed to becrystalline under a microscope. The solid was then filtered and driedusing a vacuum pump for 1 hour to give the title compound (20 mg, 63.5%yield).

Example 7 Crystalline Dioxalate Salt of Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster

150 mg of the dioxalate salt (prepared as described in Example 6) wasdissolved in 13.1 ml of 94% IPA/6% H₂O. The mixture was stirred in apre-heated 60° C. water bath for 2.5 hours. The heat was turned off andthe mixture was allowed to cool to room temperature. The vial wasrefrigerated at 4° C. After 6 days, an oily material was observed withwhat appeared to be a crystal on the side of the vial. The vial was thenallowed to reach room temperature, at which point seeds (crystallinematerial from Example 6) were added and allowed to sit for 16 days.During this time, more crystals were observed to come out of solution.The solid was then filtered and dried using a vacuum pump for 14 hoursto give the title compound (105 mg, 70% yield).

Example 8 Crystalline Freebase Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster (Form I)

109 mg of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (prepared as described in Example 1) was dissolved in 0.56 ml ofH₂O:ACN (1:1). The suspension was left in a vial (cap loosely placed ontop) to allow for a slower evaporation time. The vial was placed under anitrogen flow environment, although the nitrogen was not used forevaporation, only for the environment. A precipitate was visible within1 day, which was observed to be crystalline under a microscope. Thesolid was then placed on a high vacuum line to remove all solvent togive the title compound. Quantitative recovery, 97.8% pure by HPLC.

In an alternate procedure, after dissolving in H₂O:ACN (1:1)(approximately 350 mg/mL), the vial was stored at 5° C., and theprecipitate was visible at day 2. The solid was filtered, rinsed withwater, and dried on high vacuum overnight. Recovery was 55%, with thesolid having 98.2% purity and the liquid having 92.8% purity.

Example 9 Crystalline Freebase Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster (Form I)

50.4 mg of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (prepared as described in Example 1) was dissolved in 0.144 ml ofH₂O:ACN (1:1). The suspension was left in vial (cap loosely placed ontop) to allow for a slower evaporation time. The vial was refrigeratedat 4° C. for 6 days. A precipitate was visible after 2 days. The solidwas filtered and placed on a high vacuum line to remove all solvent andgive the title compound as a white solid (27.8 mg, 55.2% yield).

Example 10 Crystalline Freebase Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster (Form I)

230 mg of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (prepared as described in Example 1) was dissolved in 0.2 ml ofH₂O:ACN (1:1), using slight heat. The mixture was then heated in a 70°C. water bath for 2 hours. The heat was turned off and the mixture wasallowed to cool to room temperature, then refrigerated at 4° C. for 1hour. 50 μl of water was then added (oiled out), followed by theaddition of 40 μl of ACN to get the sample back into solution. Seeds(crystalline material from Example 8) were added under slow stirring atroom temperature. Crystals started to form, and the mixture was allowedto sit overnight, with slow stirring. The next day, a heat cool cyclewas applied (30° C. for 10 minutes, 40° C. for 10 minutes, then 50° C.for 20 minutes). The heat was turned off and the mixture allowed to coolovernight, with slow stirring. The next day, a second heat/cool cyclewas applied (60° C. for 1 hour, with dissolving observed at 70° C.). Theheat was turned off and the mixture allowed to cool overnight, with slowstirring. The next day, crystals were present and a third heat coolcycle was applied (60° C. for 3 hours). The heat was turned off and themixture allowed to cool overnight, with slow stirring. The next day, aheat cool cycle was applied (60° C. for 3 hours, slow cool, then 60° C.for 3 hours). The heat was turned off and the mixture allowed to coolovernight, with slow stirring. After 3 days, the solid was filtered andplaced on a high vacuum line to remove all solvent and give the titlecompound.

Example 11 Crystalline Freebase Biphenyl-2-ylcarbamic Acid1-(2-{[4-(4-Carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylEster (Form II)

70 mg of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester (prepared as described in Example 1) was dissolved in 0.1 mL ACN.After addition of 0.3 ml MTBE, the solution appeared cloudy. Anadditional 50 μl of ACN was added to clarify the solution (155 mg/mlACN:MTBE=1:2). The mixture was left in the vial and capped. Crystalsappeared by the next day. The solid was then filtered and placed on ahigh vacuum line to remove all solvent and give the title compound.

Example 12 Powder X-Ray Diffraction

Powder X-ray diffraction patterns were obtained with a Rigakudiffractometer using Cu Kα (30.0 kV, 15.0 mA) radiation. The analysiswas performed with the goniometer running in continuous-scan mode of 3°per minute with a step size of 0.03° over a range of 2 to 45°. Sampleswere prepared on quartz specimen holders as a thin layer of powderedmaterial. The instrument was calibrated with a silicon metal standard.

The PXRD pattern for a sample of the diphosphate salt of Example 2showed the material to be crystalline. A representative PXRD pattern fora sample of the crystalline diphosphate salt of Example 3 is shown inFIG. 1. The PXRD pattern for a sample of the monosulfate salt of Example4 showed the material to be crystalline. A representative PXRD patternfor a sample of the crystalline monosulfate salt of Example 5 is shownin FIG. 8. The PXRD pattern for a sample of the dioxalate salt ofExample 6 showed the material to be crystalline. A representative PXRDpattern for a sample of the crystalline dioxalate salt of Example 7 isshown in FIG. 13. The PXRD pattern for a sample of the freebase (Form I)of Examples 8 and 9 showed the material to be crystalline. Arepresentative PXRD pattern for a sample of the freebase (Form I) ofExample 10 is shown in FIG. 18. A representative PXRD pattern for asample of the freebase (Form II) of Example 11 is shown in FIG. 23.

Example 13 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-10 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Solutionssoftware. A sample of about 1-4 mg was accurately weighed into analuminum pan with lid. The sample was evaluated using a linear heatingramp of 10° C./min from ambient temperature to approximately 300° C. TheDSC cell was purged with dry nitrogen during use.

A representative DSC trace for a sample of the crystalline diphosphatesalt of Example 3 showed two transitions at about 63.8° C. and 154.3°C., as seen in FIG. 2. This DSC trace demonstrate that this crystallinediphosphate salt has acceptable to good thermal stability with themelting peak at about 154.5° C. and no thermal decomposition below 150°C. The DSC trace also showed an onset of endothermic heat flow at about135° C.

A representative DSC trace for a sample of the monosulfate salt ofExample 4 showed two transitions at about 57° C. and 73.2° C. Arepresentative DSC trace for a sample of the crystalline monosulfatesalt of Example 5 showed a transition at 76° C., as seen in FIG. 10,demonstrating that this crystalline monosulfate salt has a melting peakat about 76.5° C.

A representative DSC trace for a sample of the crystalline dioxalatesalt of Example 6 showed two transitions at 69.2° C. and 122.8° C. Arepresentative DSC trace for a sample of the crystalline dioxalate saltof Example 7 showed a transition at 73° C., as seen in FIG. 15. This DSCtrace demonstrate that this crystalline dioxalate salt has a meltingpeak at about 73.7° C.

A representative DSC trace for a sample of the crystalline freebase(Form I) of Example 8 showed a transition at 90.4° C. The DSC trace fora sample of the crystalline freebase (Form I) of Example 9 showed twotransitions at 86.1° C. and 103.6° C. A representative DSC trace for asample of the crystalline freebase of Example 10 (Form I) showed twotransitions in a closed pan (83.9° C. and 102.1° C.), but one transitionat 102.5° C. in an open pan (the early peak is due to water and/orsolvents), as seen in FIG. 19. This DSC trace demonstrate that thiscrystalline freebase has excellent thermal stability with the meltingpeak at about 102.7° C. and no thermal decomposition below 80° C. TheDSC trace also showed an onset of endothermic heat flow at about 90° C.

A representative DSC trace for a sample of the crystalline freebase(Form II) of Example 11 showed a transition at 98.6° C., as seen in FIG.24, demonstrating that this crystalline freebase has excellent thermalstability with the melting peak at about 98.6° C. and no thermaldecomposition below 75° C. The DSC trace also showed an onset ofendothermic heat flow at about 75° C.

Thermogravimetric analysis (TGA) was performed using a TA InstrumentsModel Q-50 module equipped with high resolution capability. Data werecollected and analyzed using TA Instruments Thermal Solutions software.A sample weighing about 10 mg was placed onto a platinum pan and scannedwith a high resolution-heating rate from ambient temperature to 300° C.The balance and furnace chambers were purged with nitrogen flows duringuse.

A representative TGA trace for a sample of the crystalline diphosphatesalt of Example 3 showed a loss of solvents and/or water (8.2%) attemperatures below 155° C., as seen in FIG. 3.

The TGA trace for a sample of the crystalline monosulfate salt ofExample 4 showed a loss of solvents and/or water (12.6%) at temperaturesbelow 116° C. A representative TGA trace for a sample of the crystallinemonosulfate salt of Example 5 showed a loss of solvents and/or water(13.7%) at temperatures below 150° C., as seen in FIG. 9.

The TGA trace for a sample of the crystalline dioxalate salt of Example6 showed a loss of solvents and/or water (15.4%) at temperatures below125° C. A representative TGA trace for a sample of the crystallinedioxalate salt of Example 7 showed a loss of solvents and/or water(12.7%) at temperatures below 125° C., as seen in FIG. 14.

The TGA trace for a sample of the crystalline freebase (Form I) ofExample 8 showed a loss of solvents and/or water (7.3%) at temperaturesbelow 75° C. The TGA trace for a sample of the crystalline freebase(Form I) of Example 9 showed a loss of solvents and/or water (5.2%) attemperatures below 70° C. A representative TGA trace for a sample of thecrystalline freebase (Form I) of Example 10 showed a loss of solventsand/or water (3.8%) at temperatures below 98° C., as seen in FIG. 20.

A representative TGA trace for a sample of the crystalline freebase(Form II) of Example 11 showed a loss of solvents and/or water (3.0%) attemperatures below 98° C., as seen in FIG. 25.

These TGA traces indicate that the crystalline compounds of the presentinvention lose a small amount of weight from room temperature tomoderately elevated temperatures (e.g., 75-150° C.), which is consistentwith the loss of residual moisture or solvent.

Example 14 Dynamic Moisture Sorption Assessment

A dynamic moisture sorption (DMS) assessment (also known as a moisturesorption-desorption profile) was performed using a VTI atmosphericmicrobalance, SGA-100 system (VTI Corp., Hialeah, Fla. 33016). A samplesize of approximately 10 mg was used and the humidity was set at theambient value at the start of the analysis. A typical DMS analysisconsisted of three scans: ambient to 2% relative humidity (RH), 2% RH to90% RH, 90% RH to 5% RH at a scan rate of 5% RH/step. The mass wasmeasured every two minutes and the RH was changed to the next value(+/−5% RH) when the mass of the sample was stable to within 0.01% for 5consecutive points.

A representative DMS trace for a sample of the crystalline diphosphatesalt of Example 3 showed a reversible sorption/desorption profile withlow hygroscopicity, with a 3.3% weight gain when exposed to 2-90% RH anda 0.6% weight gain in the humidity range of 40-75% RH, as shown in FIG.4.

A representative DMS trace for a sample of the crystalline monosulfatesalt of Example 5 showed a reversible sorption/desorption profile withlow hygroscopicity, with a 10% weight gain when exposed to 2-90% RH anda 1.8% weight gain in the humidity range of 40-75% RH, as shown in FIG.11.

A representative DMS trace for a sample of the crystalline dioxalatesalt of Example 7 showed a reversible sorption/desorption profile withlow hygroscopicity, with a 5.3% weight gain when exposed to 2-90% RH anda 1.1% weight gain in the humidity range of 40-75% RH, as shown in FIG.16.

A representative DMS trace for a sample of the crystalline freebase(Form I) of Example 10 showed a reversible sorption/desorption profilewith low hygroscopicity, with a 10% weight gain when exposed to 2-90% RHand a 1.2% weight gain in the humidity range of 40-75% RH, as shown inFIG. 21.

A representative DMS trace for a sample of the crystalline freebase(Form II) of Example 11 showed a reversible sorption/desorption profilewith low hygroscopicity, with a 9% weight gain when exposed to 2-90% RHand a 1.3% weight gain in the humidity range of 40-75% RH, as shown inFIG. 26.

These DMS traces demonstrate that the crystalline compounds of thepresent invention have a reversible sorption/desorption profile with lowhygroscopicity. The crystalline compounds have an acceptable weight gainwhen exposed to a broad humidity range. The reversible moisturesorption/desorption profiles demonstrate that the crystalline compoundsof the present invention possess an acceptable hygroscopicity and arenot deliquescent.

Example 15 Solid State Stability Assessment

Samples of the crystalline diphosphate salt of Example 3, about 1-2 mgeach, were stored in multiple 3 mL borosilicate vials at −20° C. (closedcontainer) and 40° C./75% RH (open and closed container). At specificintervals, the entire contents of a representative vial was analyzed bythe following HPLC method:

Column: Xterra Ms C18, 4.6×250 mm, 5 μm (Part No. 186000494); MobilePhase A: 0.1 M NH₄Ac, pH 7.0; Mobile Phase B: 100% ACN; Flow rate: 1mL/min; Injection Volume: 10 μL; Detector: 240 nm; Gradient—Time inminutes (% Mobile Phase B): 0.0 (8); 5.00 (28); 22.00 (42); 30.00 (100);35.00 (100); 35.10 (8); and 45.00 (8). Samples were prepared as 0.5mg/mL stock solutions in 10 mM citrate buffered normal saline, pH 5.

For crystalline diphosphate salt of Example 3, the initial purity of thesamples was 98.3% as determined by HPLC area percentage. After storagefor six weeks, for the samples kept under all conditions, there was nodetectable change in chemical purity, no observable change in theappearance of the material, and analysis by DSC and TGA showed nodetectable differences.

Example 16 Elemental Analysis

The following elemental percentages for samples of the crystallinecompounds of the invention were determined by combustion analysis usinga Flash EA 1112 Elemental Analyzer (CE Elantech, Lakewood, N.J.).

For the crystalline monosulfate salt of Example 5: 52.88% carbon, 7.10%hydrogen, 8.81% nitrogen, 27.17% oxygen, and 4.03% sulfur (expected);52.11% carbon, 6.90% hydrogen, 8.42% nitrogen, 24.94% oxygen, and 4.06%sulfur (results).

For the crystalline dioxalate salt of Example 7: 54.54% carbon, 6057%hydrogen, 8.15% nitrogen, and 30.74% oxygen (expected); 56.33% carbon,6.90% hydrogen, 8.22% nitrogen, and 26.32% oxygen (results).

Example 17 Micronization

A 13 g sample of the crystalline diphosphate salt of Example 3 wasmicronized with a jet mill to give 8.7 g of a free-flowing white powderwith birefringence observed upon microscopic examination (67% recovery).Pre-micronization, the crystalline diphosphate had an initial purity of98.1% as determined by HPLC area percentage. The purity of themicronized material was the same. The water content of thepre-micronized material was 6.54 wt %, and the water content of themicronized material was 6.23 wt %.

No issues were encountered during the micronization process. Particlesize distribution was as follows:

Pre-Micronization Post- Micronization D (v, 0.9) 38.6 μm  5.2 μm D (v,0.5) 9.9 μm 2.2 μm D (v, 0.1) 1.7 μm 0.4 μm

No significant changes were observed in the powder x-ray diffractionpattern, TGA, DSC, DMS, chemical purity, chiral purity and moisturecontent for the micronized material compared to the unmicronizedmaterial. For example, as noted in Example 14, a representative DMStrace for a sample of the crystalline diphosphate salt of Example 3showed a 0.6% weight gain in the humidity range of 40-75% RH, while themicronized material showed a 0.7% weight gain in this humidity range.

Example 18 Inhalation Solution Stability

A solution was prepared with 0.5 mg/mL freebase equivalents (using acrystalline diphosphate salt prepared as described in Example 3) in 10mM citrate buffered normal saline, pH 5. The solubility of thecrystalline salt was greater than 40 mg/mL of freebase equivalent in thebuffer. Less than 0.5% degradation was observed after storage for onemonth at 40° C./75% RH.

Assay 1 Radioligand Binding Assay Membrane Preparation from CellsExpressing hM₁, hM₂, hM₃ and hM₄ Muscarinic Receptor Subtypes

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in medium consisting of HAM's F-12 supplemented with 10% FBSand 250 μg/mL Geneticin. The cells were grown in a 5% CO₂, 37° C.incubator and lifted with 2 mM EDTA in dPBS. Cells were collected by 5minute centrifugation at 650×g, and cell pellets were either storedfrozen at −80° C. or membranes were prepared immediately. For membranepreparation, cell pellets were resuspended in lysis buffer andhomogenized with a Polytron PT-2100 tissue disrupter (Kinematica AG; 20seconds×2 bursts). Crude membranes were centrifuged at 40,000×g for 15minutes at 4° C. The membrane pellet was then resuspended withresuspension buffer and homogenized again with the Polytron tissuedisrupter. The protein concentration of the membrane suspension wasdetermined by the method described in Lowry, O. et al., Journal ofBiochemistry 193:265 (1951). All membranes were stored frozen inaliquots at −80° C. or used immediately. Aliquots of prepared hM₅receptor membranes were purchased directly from Perkin Elmer and storedat −80° C. until use.

Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂, hM₃,hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 100 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM4 or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10-15 μg for hM₂, 10-20 μg for hM₃, 10-20 ngfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 nM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 pM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer Inc., Wellesley, Mass.) pre-treated in 1%BSA. Filter plates were rinsed three times with wash buffer (10 mMHEPES) to remove unbound radioactivity. Plates were then air dried, and50 μL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.,Wellesley, Mass.) was added to each well. The plates were then countedin a PerkinElmer Topcount liquid scintillation counter (PerkinElmerInc., Wellesley, Mass.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the one-site competition model.K_(i) values for test compounds were calculated from observed IC₅₀values and the K_(D) value of the radioligand using the Cheng-Prusoffequation (Cheng Y; Prusoff W. H. Biochemical Pharmacology22(23):3099-108 (1973)). K_(i) values were converted to pK_(i) values todetermine the geometric mean and 95% confidence intervals. These summarystatistics were then converted back to K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. The compound offormula I was found to have a K_(i) value of less than about 5 nM forthe M₃ muscarinic receptor subtype when tested in this or a similarassay.

Assay 2 Muscarinic Receptor Functional Potency Assays Blockade ofAgonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound was determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO—K1 cells expressing the hM₂ receptor.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells were rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in the Cell Culture andMembrane Preparation section above. The detached cells were washed twiceby centrifugation at 650×g for five minutes in 50 mLs dPBS. The cellpellet was then re-suspended in 10 mL dPBS, and the cells were countedwith a Coulter Z1 Dual Particle Counter (Beckman Coulter, Fullerton,Calif.). The cells were centrifuged again at 650×g for five minutes andre-suspended in stimulation buffer to an assay concentration of1.6×10⁶-2.8×10⁶ cells/mL. The test compound was initially dissolved to aconcentration of 400 μM in dilution buffer (dPBS supplemented with 1mg/mL BSA (0.1%)), and then serially diluted with dilution buffer tofinal molar concentrations ranging from 100:M to 0.1 nM. Oxotremorinewas diluted in a similar manner.

To measure oxotremorine inhibition of AC activity, 25 μL forskolin (25μM final concentration diluted in dPBS), 25 μL diluted oxotremorine, and50 μL cells were added to agonist assay wells. To measure the ability ofa test compound to block oxotremorine-inhibited AC activity, 25 μLforskolin and oxotremorine (25 μM and 5 μM final concentrations,respectively, diluted in dPBS) 25 μL diluted test compound, and 50 μLcells were added to remaining assay wells.

Reactions were incubated for 10 minutes at 37° C. and stopped byaddition of 100 μL ice-cold detection buffer. Plates were sealed,incubated overnight at room temperature and counted the next morning ona PerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) wascalculated based on the counts observed for the samples and cAMPstandards, as described in the manufacturer's user manual. Data wereanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the EC₅₀ of theoxotremorine concentration-response curve and the oxotremorine assayconcentration as the K_(D) and [L], respectively. The K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The compound offormula I was found to have a K_(i) value of less than about 5 nM forblockade of oxotremorine-inhibition of forskolin-mediated cAMPaccumulation in CHO—K1 cells expressing the hM₂ receptor, when tested inthis or a similar assay.

Blockade of Agonist-Mediated [³⁵S]GTPγS-Binding

In a second functional assay, the functional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS-binding in CHO—K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes were thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes were briefly homogenized using a PolytronPT-2100 tissue disrupter and then added to the assay plates. The EC₉₀value (effective concentration for 90% maximal response) for stimulationof [³⁵S]GTPγS binding by the agonist oxotremorine was determined in eachexperiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following was added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine (EC₉₀) and GDP (3 μM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates were then incubated at 37° C. for 60 minutes. The assayplates were filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates were rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) was added to each well, and each plate wassealed and radioactivity counted on a topcounter (PerkinElmer). Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The compound offormula I was found to have a K_(i) value of less than about 5 nM forblockade of oxotremorine-stimulated [³⁵S]GTPγS-binding in CHO—K1 cellsexpressing the hM₂ receptor, when tested in this or a similar assay.

Blockade of Agonist-Mediated Calcium Release Via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM_(i), hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in HBSS without calcium andmagnesium) to remove growth media and leaving 50 μL/well of FLIPRbuffer. The cells are then incubated with 50 μL/well of 4 μM FLUO-4AM (a2× solution was made) for 40 minutes at 37° C., 5% carbon dioxide.Following the dye incubation period, cells are washed two times withFLIPR buffer, leaving a final volume of 50 μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F)^1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)is prepared in stimulation plates such that an EC %) concentration ofoxotremorine is added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence is expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data is analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values aredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The compound offormula I was found to have a K_(i) value of less than about 5 nM forblockade of agonist-mediated calcium release in CHO cells stablyexpressing the hM₃ receptor, when tested in this or a similar assay.

Assay 3 Determination of Duration of Bronchoprotection in Guinea PigModel of Acetylcholine-Induced Bronchoconstriction

This in vivo assay is used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.Groups of six male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g are individuallyidentified by cage cards. Throughout the study animals are allowedaccess to food and water ad libitum.

Test compounds are administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers are arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Guinea pigsare exposed to an aerosol of a test compound or vehicle (WFI). Theseaerosols are generated from aqueous solutions using an LC Star NebulizerSet (Model 22F51, PARI Respiratory Equipment, Inc. Midlothian, Va.)driven by a mixture of gases (CO₂=5%, O₂=21% and N₂=74%) at a pressureof 22 psi. The gas flow through the nebulizer at this operating pressureis approximately 3 L/minute. The generated aerosols are driven into thechambers by positive pressure. No dilution air is used during thedelivery of aerosolized solutions. During the 10 minute nebulization,approximately 1.8 mL of solution is nebulized. This is measuredgravimetrically by comparing pre- and post-nebulization weights of thefilled nebulizer.

The bronchoprotective effects of test compounds administered viainhalation are evaluated using whole body plethysmography at 1.5, 24, 48and 72 hours post-dose.

Forty-five minutes prior to the start of the pulmonary evaluation, eachguinea pig is anesthetized with an intramuscular injection of ketamine(43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg).After the surgical site is shaved and cleaned with 70% alcohol, a 2-3 cmmidline incision of the ventral aspect of the neck was made. Then, thejugular vein is isolated and cannulated with a saline-filledpolyethylene catheter (PE-50, Becton Dickinson, Sparks, Md.) to allowfor intravenous infusions of ACh (Sigma-Aldrich, St. Louis, Mo.) insaline. The trachea is then dissected free and cannulated with a 14Gteflon tube (#NE-014, Small Parts, Miami Lakes, Fla.). If required,anesthesia is maintained by additional intramuscular injections of theaforementioned anesthetic mixture. The depth of anesthesia is monitoredand adjusted if the animal responds to pinching of its paw or if therespiration rate is greater than 100 breaths/minute.

Once the cannulations are complete, the animal is placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) isinserted to measure pulmonary driving pressure (pressure). The teflontracheal tube is attached to the opening of the plethysmograph to allowthe guinea pig to breathe room air from outside the chamber. The chamberis then sealed. A heating lamp is used to maintain body temperature andthe guinea pig's lungs are inflated 3 times with 4 mL of air using a 10mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City, Mo.) toensure that the lower airways do not collapse and that the animal doesnot suffer from hyperventilation.

Once it is determined that baseline values are within the range 0.3-0.9mL/cm H₂O for compliance and within the range 0.1-0.199 cm H₂O/mL persecond for resistance, the pulmonary evaluation is initiated. A Buxcopulmonary measurement computer program enables the collection andderivation of pulmonary values.

Starting this program initiates the experimental protocol and datacollection. The changes in volume over time that occur within theplethysmograph with each breath are measured via a Buxco pressuretransducer. By integrating this signal over time, a measurement of flowis calculated for each breath. This signal, together with the pulmonarydriving pressure changes, which are collected using a Sensym pressuretransducer (#TRD4100), is connected via a Buxco (MAX 2270) preamplifierto a data collection interface (#'s SFT3400 and SFT3813). All otherpulmonary parameters are derived from these two inputs.

Baseline values are collected for 5 minutes, after which time the guineapigs are challenged with ACh. ACh (0.1 mg/mL) is infused intravenouslyfor 1 minute from a syringe pump (sp210iw, World Precision Instruments,Inc., Sarasota, Fla.) at the following doses and prescribed times fromthe start of the experiment: 1.9 μg/minute at 5 minutes, 3.8 μg/minuteat 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at 20minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes. Ifresistance or compliance has not returned to baseline values at 3minutes following each ACh dose, the guinea pig's lungs are inflated 3times with 4 mL of air from a 10 mL calibration syringe. Recordedpulmonary parameters includes respiration frequency (breaths/minute),compliance (mL/cm H₂O) and pulmonary resistance (cm H₂O/mL per second).Once the pulmonary function measurements are completed at minute 35 ofthis protocol, the guinea pig is removed from the plethysmograph andeuthanized by carbon dioxide asphyxiation.

The data are evaluated in one or both of the following ways:

(a) Pulmonary resistance (R_(L), cm H₂O/mL per second) is calculatedfrom the ratio of “change in pressure” to “the change in flow.” TheR_(L) response to ACh (60 μg/min, 1H) is computed for the vehicle andthe test compound groups. The mean ACh response in vehicle-treatedanimals, at each pre-treatment time, is calculated and used to compute %inhibition of ACh response, at the corresponding pre-treatment time, ateach test compound dose Inhibition dose-response curves for ‘R_(L)’ arefitted with a four parameter logistic equation using GraphPad Prism,version 3.00 for Windows (GraphPad Software, San Diego, Calif.) toestimate bronchoprotective ID₅₀ (dose required to inhibit the ACh (60μg/min) bronchoconstrictor response by 50%). The equation used is asfollows:Y=Min+(Max−Min)/(1+10^(((log ID50-X)*Hillslope)))where X is the logarithm of dose, Y is the response (% Inhibition of AChinduced increase in R_(L)). Y starts at Min and approachesasymptotically to Max with a sigmoidal shape.

(b) The quantity PD₂, which is defined as the amount of ACh or histamineneeded to cause a doubling of the baseline pulmonary resistance, iscalculated using the pulmonary resistance values derived from the flowand the pressure over a range of ACh or histamine challenges using thefollowing equation (which is derived from a equation used to calculatePC₂₀ values described in American Thoracic Society. Guidelines formethacholine and exercise challenge testing—1999. Am J Respir Grit CareMed. 161: 309-329 (2000)):

${PD}_{2} = {{antilog}\left\lbrack {{\log\; C_{1}} + \frac{\left( {{\log\; C_{2}} - {\log\; C_{1}}} \right)\left( {{2\; R_{0}} - R_{1}} \right)}{R_{2} - R_{1}}} \right\rbrack}$where: C₁ is the concentration of ACh or histamine preceding C₂; C₂ isthe concentration of ACh or histamine resulting in at least a 2-foldincrease in pulmonary resistance (R_(L)); R₀ is the baseline R_(L)value; R₁ is the R_(L) value after C₁; and R₂ is the R_(L) value afterC₂. An efficacious dose is defined as a dose that limits thebronchrestriction response to a 50 μg/mL dose of ACh to a doubling ofthe baseline pulmonary resistance (PD₂₍₅₀₎).

Statistical analysis of the data is performed using a two-tailedStudents t-test. A P-value<0.05 is considered significant. Generally,test compounds having a PD₂₍₅₀₎ less than about 200 μg/mL forACh-induced bronchoconstriction at 1.5 hours post-dose in this assay arepreferred. The compound of formula I is expected to have a PD₂₍₅₀₎ lessthan about 200 μg/mL for ACh-induced bronchoconstriction at 1.5 hourspost-dose, when tested in this or a similar assay.

Assay 4 Inhalation Guinea Pig Salivation Assay

Guinea pigs (Charles River, Wilmington, Mass.) weighing 200-350 g areacclimated to the in-house guinea pig colony for at least 3 daysfollowing arrival. Test compound or vehicle are dosed via inhalation(1H) over a 10 minute time period in a pie shaped dosing chamber (R&SMolds, San Carlos, Calif.). Test solutions are dissolved in sterilewater and delivered using a nebulizer filled with 5.0 mL of dosingsolution. Guinea pigs are restrained in the inhalation chamber for 30minutes. During this time, guinea pigs are restricted to an area ofapproximately 110 sq. cm. This space is adequate for the animals to turnfreely, reposition themselves, and allow for grooming. Following 20minutes of acclimation, guinea pigs are exposed to an aerosol generatedfrom a LS Star Nebulizer Set (Model 22F51, PARI Respiratory Equipment,Inc. Midlothian, Va.) driven by house air at a pressure of 22 psi. Uponcompletion of nebulization, guinea pigs are evaluated at 1.5, 6, 12, 24,48, or 72 hrs after treatment.

Guinea pigs are anesthetized one hour before testing with anintramuscular (IM) injection of a mixture of ketamine 43.75 mg/kg,xylazine 3.5 mg/kg, and acepromazine 1.05 mg/kg at an 0.88 mL/kg volumeAnimals are placed ventral side up on a heated (37° C.) blanket at a 20degree incline with their head in a downward slope. A 4-ply 2×2 inchgauze pad (Nu-Gauze General-use sponges, Johnson and Johnson, Arlington,Tex.) is inserted in the guinea pig's mouth. Five minutes later, themuscarinic agonist pilocarpine (3.0 mg/kg, SC) is administered and thegauze pad is immediately discarded and replaced by a new pre-weighedgauze pad. Saliva is collected for 10 minutes, at which point the gauzepad is weighed and the difference in weight recorded to determine theamount of accumulated saliva (in mg). The mean amount of salivacollected for animals receiving the vehicle and each dose of testcompound is calculated. The vehicle group mean is considered to be 100%salivation. Results are calculated using result means (n=3 or greater).Confidence intervals (95%) are calculated for each dose at each timepoint using two-way ANOVA. This model is a modified version of theprocedure described in Rechter, “Estimation of anticholinergic drugeffects in mice by antagonism against pilocarpine-induced salivation”Ata Pharmacol Toxicol 24:243-254 (1996).

The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, is calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose. Theinhibition dose-response data are fitted to a four parameter logisticequation using GraphPad Prism, version 3.00 for Windows (GraphPadSoftware, San Diego, Calif.) to estimate anti-sialagogue ID₅₀ (doserequired to inhibit 50% of pilocarpine-evoked salivation). The followingequation is used:Y=Min+(Max−Min)/(1+10^(((log ID50-X)*Hillslope)))where X is the logarithm of dose, Y is the response (% inhibition ofsalivation). Y starts at Min and approaches asymptotically to Max with asigmoidal shape.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ is usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred. The compound of formula I is expected tohave an apparent lung-selectivity index greater than about 5, whentested in this or a similar assay.

Assay 5 Methacholine-Induced Depressor Responses in Conscious GuineaPigs

Healthy, adult, male Sprague-Dawley guinea pigs (Harlan, Indianapolis,Ind.), weighing between 200 and 300 g are used in these studies. Underisoflurane anesthesia (to effect), animals are instrumented with commoncarotid artery and jugular vein catheters (PE-50 tubing). The cathetersare exteriorized utilizing a subcutaneous tunnel to the subscapulararea. All surgical incisions are sutured with 4-0 Ethicon Silk and thecatheters locked with heparin (1000 units/mL). Each animal isadministered saline (3 mL, SC) at the end of surgery as well asbuprenorphine (0.05 mg/kg, IM) Animals are allowed to recover on aheating pad before being returned to their holding rooms.

Approximately 18 to 20 hours following surgery, the animals are weighedand the carotid artery catheter on each animal is connected to atransducer for recording arterial pressure. Arterial pressure and heartrate are recorded using a Biopac MP-100 Acquisition System. Animals areallowed to acclimate and stabilize for a period of 20 minutes.

Each animal is challenged with MCh (0.3 mg/kg, IV) administered throughthe jugular venous line and the cardiovascular response is monitored for10 minutes. The animals are then placed into the whole body dosingchamber, which is connected to a nebulizer containing the test compoundor vehicle solution. The solution is nebulized for 10 minutes using agas mixture of breathable air and 5% carbon dioxide with a flow rate of3 liters/minute. The animals are then removed from the whole bodychamber and returned to their respective cages. At 1.5 and 24 hourspost-dosing, the animals are re-challenged with MCh (0.3 mg/kg, IV) andthe hemodynamic response is determined. Thereafter, the animals areeuthanized with sodium pentobarbital (150 mg/kg, IV).

MCh produces a decrease in mean arterial pressure (MAP) and decrease inheart rate (bradycardia). The peak decrease, from baseline, in MAP(depressor responses) is measured for each MCh challenge (before andafter 1H dosing). The effects of treatment on the MCh responses areexpressed as % inhibition (mean+/−SEM) of the control depressorresponses. Two-way ANOVA with the appropriate post-hoc test is used totest the effects of treatment and pre-treatment time. The depressorresponses to MCh are expected to be relatively unchanged at 1.5 and 24hours after inhalation dosing with vehicle.

The ratio of the anti-depressor ID₅₀ to bronchoprotective ID₅₀ is usedto compute apparent lung-selectivity of the test compound. Generally,compounds having an apparent lung-selectivity index greater than 5 arepreferred. The compound of formula I is expected to have an apparentlung-selectivity index greater than 5, when tested in this or a similarassay.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

What is claimed is:
 1. A crystalline dioxalate salt ofbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester, characterized by a powder x-ray diffraction pattern comprisingdiffraction peaks at 2θ values of 8.7±0.2, 14.0±0.2, 19.4±0.2, and22.9±0.2.
 2. The crystalline dioxalate salt of claim 1, wherein thepowder x-ray diffraction pattern further comprises diffraction peaks at2θ values selected from 7.7±0.2, 13.5±0.2, 14.8±0.2, 15.4±0.2, 15.8±0.2,23.3±0.2, and 24.6±0.2.
 3. The crystalline dioxalate salt of claim 1,characterized by a powder x-ray diffraction pattern in which the peakpositions are substantially in accordance with the peak positions of thepattern shown in FIG.
 13. 4. The crystalline dioxalate salt of claim 1,characterized by a differential scanning calorimetry trace which shows amaximum endothermic heat flow at about 73.7° C.
 5. The crystallinedioxalate salt of claim 1, characterized by a differential scanningcalorimetry trace substantially in accordance with that shown in FIG.15.
 6. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and the crystalline dioxalate salt of claim
 1. 7. Thecomposition of claim 6, wherein the composition is formulated foradministration by inhalation.
 8. The composition of claim 6, wherein thecarrier is an aqueous isotonic saline solution having a pH in the rangeof from about 4 to about
 6. 9. The composition of claim 8, whichcomprises a citrate buffer.
 10. A drug delivery device comprising a drypowder inhaler containing a pharmaceutical composition comprising apharmaceutically acceptable carrier and the crystalline dioxalate saltof claim
 1. 11. The crystalline dioxalate salt of claim 1, in micronizedform.
 12. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and the crystalline dioxalate salt of claim 1, inmicronized form.
 13. The composition of claim 12, wherein the carrier islactose.
 14. A process for preparing the crystalline dioxalate salt ofclaim 1, comprising: forming a seed crystal of a crystalline dioxalatesalt of biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester by contacting biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester with oxalic acid; forming a dioxalate salt ofbiphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester by contacting biphenyl-2-ylcarbamic acid1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-ylester with oxalic acid, and dissolving the salt in an inert diluent toform a solution; and adding the seed crystal to the solution.
 15. Amethod for treating a pulmonary disorder, the method comprisingadministering to a patient a therapeutically effective amount of thecrystalline dioxalate of claim
 1. 16. A method of producingbronchodilation comprising administering to a patient by inhalation, abronchodilation-producing amount of the crystalline dioxalate salt ofclaim
 1. 17. A method of treating chronic obstructive pulmonary diseaseor asthma, comprising administering to a patient a therapeuticallyeffective amount of the crystalline dioxalate salt of claim 1.